Streptococcus pneumoniae (S. pneumoniae) and nontypeable Haemophilus influenzae (NTHi) are the major human pathogens causing otitis media (OM). Inflammation is the hallmark of OM. However, the molecular mechanisms underlying inflammation in OM remain poorly defined. Our long-term objective is to understand how the inflammatory responses are induced and regulated in the pathogenesis of OM. Given the fact that, in in vivo situations, multiple factors are existing simultaneously and up to 24-48% of OM patients have combined infections with S. pneumoniae and NTHi, we hypothesize that NF-kB is synergistically regulated by multiple pathogenic inducers via activation of multiple signaling pathways in the pathogenesis of OM (overall hypothesis). Indeed, our preliminary results indicate that NTHi and S. pneumoniae or NTHi and TNF-alfa synergistically activate NF-kB and cytokine production via multiple signaling pathways involving IKBcz, p38 MAPK and MEKK1. Thus, these encouraging results have laid a solid foundation for further investigation of the signaling mechanisms underlying NF-kB activation and cytokine production by multiple pathogenic inducers (short-term objective). Aim 1. Determine whether and how activation of TGF- beta-Smad signaling pathway is required for NTHi-induced NF-kB activation and cytokine production by perturbing their signaling. Aim 2. Determine the signaling mechanisms underlying the synergistic activation of NF-KB and cytokine induction by S. pneumoniae and NTHi by perturbing IKBa, p38 MAPK and TGF-B-Smad signaling. Aim 3. Determine the signaling mechanisms underlying the synergistic activation of NF-kB and cytokine induction by TNF-a and NTHi by perturbing IkBa, p 38 and MEKK1 signaling. Significance: Investigating the signaling mechanisms underlying the combinatorial regulation of NF-kB by multiple pathogenic factors will not only bring new insights into the regulation of inflammation in the pathogenesis of OM, but also open up novel therapeutic targets for inhibiting inflammatory responses in patients with OM.